Dysregulated Lipid Metabolism as a Central Driver of Atherosclerotic Plaque Pathology

Abstract

It has long been recognized that elevated circulating lipid levels are among the strongest risk factors for the development of plaques within the arterial wall that are characteristic of atherosclerotic cardiovascular disease. Indeed, decades of studies have identified the deposition of low-density lipoprotein as an initiator of this disease, which coordinates the vascular and immune dysfunction that fuels the advancement of the atherosclerotic plaque. However, in the vessel wall, deposited cholesterol and fatty acids are dynamic in nature and engage signaling pathways. Shifting from metabolic-related pathways, lipid modifications and their conversion to intermediates engage signaling cascades that further perpetuate the inflammatory milieu of the atherosclerotic plaque and its progression towards the fatal end-stage events associated with cardiovascular disease, including myocardial infarction. In this review, we will cover the cellular and molecular mechanisms that preserve homeostasis and advance disease, including how lipid species induce endothelial dysfunction and drive the development of macrophage foam cells. We will additionally discuss ongoing therapeutic strategies to combat the hyperlipidemia that underlies atherogenesis.

1. Introduction

Lipids, including triglycerides, phospholipids, and cholesterol, are a diverse set of macromolecules that play important roles in preserving cellular homeostasis [1,2,3]. Their production, transport, and metabolism are all involved in critical biological processes, such as production of adenosine triphosphate (ATP) for energy, components of cell membranes, synthesis of hormones, and cell signaling, among many other functions. However, when these processes go awry, diseases such as atherosclerotic cardiovascular disease (ASCVD) develop. Otherwise referred to as ischemic heart disease or coronary artery disease (CAD), ASCVD is a complication of cardiovascular disease (CVD) and remains the number one cause of mortality worldwide [4,5,6]. This disease can occur sub-clinically for several decades as lipids deposit within the arterial wall to form the plaques characteristic of ASCVD. As the plaque advances through sequential stages, it becomes vulnerable and prone to rupture or erosion, leading to major adverse cardiovascular events (MACE), including stroke or myocardial infarction [7,8,9,10]. Traditional risk factors for ASCVD include age, genetics, diabetes and other metabolic diseases, hypertension, smoking, and elevated low-density lipoprotein (LDL) cholesterol [11]. Apart from simple accumulation in the artery, evolving studies have described the critical influences of lipoprotein and lipid subtypes in signaling and the advancement of the plaque towards these end-stage complications. In this review, we will cover the production and transport of lipids and their contribution within the arterial wall towards driving atherosclerotic plaque pathology. We will also highlight existing and new lipid-based therapeutic targets for the treatment of ASCVD.

2. Hepatic Synthesis, Composition, and Transport of Circulating Lipoproteins

Lipoproteins are lipid transport molecules that support the movement of hydrophobic lipids through the circulation to peripheral tissues [1]. Lipoproteins include chylomicrons, very low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), low-density lipoprotein (LDL), and high-density lipoprotein (HDL). While IDL, LDL, and HDL are considered cholesterol-rich lipoproteins, chylomicrons and VLDL are triglyceride-rich lipoproteins (TRLs). These lipoproteins are produced by the liver, except for chylomicrons, which are assembled in the small intestine. In addition to their major lipid species and origin, lipoproteins are classified based on several parameters, including size and density, which have been reviewed extensively elsewhere [2,12]. VLDL composition includes various apolipoproteins embedded in the phospholipid monolayer, such as apolipoprotein-B100 and -C3 (Apo-B100 and Apo-C3, respectively), which serve as mediators of lipoprotein biology [13,14]. Tissue metabolism of VLDL by the adipose tissue and skeletal muscle is mediated by lipoprotein, hepatic, and endothelial lipases, which liberate triglycerides. Removal of these triglycerides from VLDL particles can then form IDL and LDL [1,13,14]. Additionally, hydrolysis of TRLs by lipoprotein lipase (LPL) decreases particle size, allowing them to more easily cross the endothelium, where they can be retained as part of the atherosclerotic plaque [15,16,17].

LDL cholesterol was first recognized as a major driver of atherosclerosis in 1955 [18,19,20]. Synthesis of LDL by the liver includes one Apo-B100 per LDL particle, which is a glycoprotein synthesized and secreted by the liver [2]. Colloquially referred to as the “bad cholesterol”, LDL is responsible for the transport of cholesterol from the liver to peripheral tissues, including the arterial wall. LDL in circulation is cleared by the liver via binding of Apo-B100 on the LDL particle to low-density lipoprotein receptor (LDLR) in the liver. Upon binding, LDL-LDLR is internalized by hepatocytes via receptor-mediated endocytosis, where LDL is trafficked to the lysosome for degradation [20,21]. Subsequently, LDLR is returned to the plasma membrane to promote further clearance of LDL from the circulation. Despite being cloned and sequenced in the mid-1980s, the structure of Apo-B100 on LDL and interactions with LDLR were only described in 2024 [22,23,24,25]. These interactions of Apo-B100 with LDLR are critical for the uptake and clearance of LDL cholesterol from the circulation and therefore may open further therapeutic avenues for patients who require more aggressive lipid-lowering.

HDL, on the other hand, is often referred to as the “good cholesterol”. The major apolipoprotein on HDL is Apo-A1. Unlike LDL, which carries only one Apo-B100, there are multiple Apo-A1 per HDL particle [2,26]. HDL has been found to have many roles that participate in atherogenesis, which are reviewed elsewhere [27]. The role of HDL as the “good cholesterol” in atherosclerosis relates to reverse cholesterol transport (RCT), which begins in the plaque where Apo-A1 and HDL receive cholesterol via cholesterol efflux, followed by transport to the liver [28]. Apo-A1 on HDL subsequently binds to scavenger receptor B1 (SR-B1) expressed on hepatocytes, which then engulf the HDL particle. Considered the rate-limiting enzyme of RCT, cholesterol 7 alpha-hydroxylase (CYP7A1) converts cholesterol to bile acids, and its efflux is mediated by obligate heterodimers of adenosine triphosphate-binding cassette transporter G5/G8 (ABCG5/8). This ultimately leads to the excretion of cholesterol in the feces via the intestine. In fact, a recent systems biology approach using metabolomics and transcriptomics in pig models of atherosclerosis has identified elevations in bile acids in driving inter-organ dysfunction leading to atherogenesis [29].

Given the importance of these lipoproteins in the development of atherosclerosis, levels of these lipid species have been established as a diagnostic measure in assessing ASCVD risk (

Table 1. Clinical values for circulating lipids in adults ¹.

Circulating Lipids	Normal/Optimal Ranges for Adults
LDL cholesterol	<100 mg/dL (2.6 mM)
HDL cholesterol	>40 mg/dL (1.0 mM)
Fasting triglycerides	<150 mg/dL (1.7 mM)
Lp(a) *	<30 mg/dL (62 nM)

¹ Data compiled from [11,32,33,34]. * Need for standardization in Lp(a) measurements contribute to the variability in recommended values [35]. Concentrations calculated according to [36].

). Of clinical importance, familial hypercholesterolemia (FH) is characterized by chronic elevations in LDL cholesterol, which affects approximately 1 in 220 people worldwide [30,31]. This condition is underscored by genetic variants in LDLR, Apo-B, or gain-of-function mutations in proprotein convertase subtilisin/kexin type 9 (PCSK9), all of which function to prevent the clearance of LDL cholesterol from the circulation.

3. Lipid-Dependent Aortic Endothelial Dysfunction and Monocyte Entry

Lipid-mediated dysfunction, as one of the central drivers of ASCVD, is captured by three prevailing hypotheses. The first hypothesis put forth by Ross and Glomset is the response-to-injury hypothesis, which states that endothelial injury due to hyperlipidemia, among other insults, initiates lipid accumulation in the tissue and subsequently the inflammation that drives atherosclerosis [37,38]. Secondly, the cumulative exposure hypothesis postulates that modified LDL becomes trapped within the intima of the artery, causing initial plaque development, which in turn increases shear stress and mechanical burden of the vessel wall [39]. Through continuous LDL accumulation, there is further dysfunction of the plaque that drives progression towards MACE. The third hypothesis is the lipid retention hypothesis, whereby excess lipoproteins cross the endothelium, enter the arterial wall, and are then subject to modifications, such as oxidation, leading to oxidized low-density lipoprotein (oxLDL) or aggregation, yielding aggregated low-density lipoprotein (agLDL) [40]. These modified LDLs subsequently induce the formation of macrophage foam cells, discussed below, which in turn lead to the cooperativity of lipids and inflammation that potentiate plaque development [41]. Below, we discuss the direct effects that occur in response to lipids, where the effects of disturbed flow secondary to plaque growth and feedback mechanisms that further induce atherogenic signaling have been reviewed extensively elsewhere [42,43].

Owing to its role in vascular surveillance and providing a physical barrier between the circulation and the tissue, it is unsurprising that a unifying mechanism in the development of ASCVD is changes to the endothelium, including its activation, dysfunction, and mesenchymal transition [44,45]. Briefly, endothelial activation involves the increased expression of pro-inflammatory adhesion molecules, cytokines, and chemokines. Endothelial dysfunction is characterized by impaired nitric oxide signaling and redox imbalance. Atherogenic lipids can also cause endothelial cells to adopt a mesenchymal phenotype, termed endothelial-to-mesenchymal transition (EndMT) [46]. However, these endothelial defects do not occur as separate processes. For example, nitric oxide itself has been shown to repress inflammatory signaling in part by blocking NF-κB signaling through S-nitrosylation of NF-κB subunits or inhibitory κB kinase [47,48,49,50]. Nevertheless, this results in blunted downstream activation of endothelial cells, including production of inflammatory cytokines and adhesion molecules, as described below.

One of the hallmarks of a dysfunctional endothelium is increased permeability due to structural changes that compromise barrier integrity [51]. In fact, hypercholesterolemia and atherogenic lipids have been shown to inhibit the production of nitric oxide from endothelial cells via promoting inhibitory interactions in the plasma membrane between caveolin and the enzyme that produces nitric oxide, endothelial nitric oxide synthase, thereby impairing endothelial function [52,53,54,55]. Passive diffusion has long been touted as the mechanism by which circulating LDL can enter the artery due to increased endothelial permeability [56]. However, more recent studies have challenged this notion and proposed an alternative mechanism for LDL transport. Known as transcytosis, LDL is transported from the lumen to the intima across the endothelial cells. There are two forms of transcytosis, fluid-phase and receptor-mediated transcytosis [57,58]. In the former, caveolae facilitate the uptake of LDL at the cell surface, whereas in the latter, LDL uptake is mediated by cell surface receptors, including LDLR and SR-B1. Of note, expression of SR-B1 and its partner DOCK4 (dedicator of cytokinesis 4) have been found to be increased in mouse and human atherosclerosis-prone aortic regions [59]. Interestingly, as an additional anti-atherogenic function, Apo-A1 on HDL competes with LDL for SR-B1 binding and thus reduces the transcytosis of LDL across the endothelium and its subsequent accumulation in the aorta [60].

Endothelial cell activation involves pro-inflammatory processes that promote leukocyte extravasation into the plaque (Figure 1). Common to these pro-inflammatory mechanisms that drive atherogenesis, oxLDL, which is a prominent modified LDL particle found in human atheromas, activates the inflammatory transcription factor NF-κB in macrophages as well as endothelial cells [61,62,63]. OxLDL also increases expression of one of its receptors, lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1, also known as OLR1), to promote NF-κB nuclear translocation and subsequent pro-inflammatory signaling [64,65,66]. One of the key lipid-induced pro-inflammatory processes of endothelial cells that drives atherogenesis is the induction of selectins, such as P- and E-selectin, that slow leukocyte rolling along the endothelium [67,68,69,70,71,72,73]. Furthermore, endothelial cells also upregulate adhesion molecules, including vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1), although studies in mice suggest a greater importance of VCAM-1, particularly in early atherosclerosis [74,75,76,77,78,79]. These adhesion molecules, their respective ligands, and signaling pathways have been reviewed extensively elsewhere [67,73,80]. Importantly, upregulation of these factors on endothelial cells induced by oxLDL and LOX-1 are reversed by cholesterol-lowering treatment (statins) [81].

Of importance to plaque development, observations in mice and humans have robustly demonstrated increased circulating monocytes during the hyperlipidemia that underscores ASCVD [82,83,84,85]. Broadly, monocytes are divided into three subsets: classical (Ly6C^hi in mice, CD14⁺ CD16⁻ in humans), intermediate (Ly6C^int in mice, CD14⁺ CD16⁺ in humans), and nonclassical monocytes (Ly6Clo in mice, CD14^dim CD16⁺ in humans) [86,87,88,89]. In particular, hyperlipidemia favors the production of classical monocytes, which are pro-inflammatory in nature. Observations in mice demonstrate that hypercholesterolemia supports the survival and proliferation of these classical monocytes, which is reversed upon treatment with statins [90]. Furthermore, the spleen is an important reservoir for the production of classical monocytes during atherosclerosis [91]. This is correlated with increased splenic activity as determined by functional imaging in patients with CVD, which was used as a prognostic indicator for future MACE [92]. In addition to their production, hypercholesterolemia also promotes the chemotaxis of monocytes towards the injured vessel wall in part due to an upregulation of chemokine receptors, such as C-C motif chemokine receptor 2 (CCR2) [93,94,95]. Mechanistic studies demonstrate that in response to oxLDL, endothelial cells produce chemokines to recruit these monocytes, including monocyte chemotactic protein 1 (MCP-1, also known as chemokine (C-C motif) ligand 2 [CCL2]) and interleukin-8 (IL-8, also known as CXCL8) [96,97,98,99,100]. Specifically, deletion of these factors or their cognate receptors demonstrates their roles in driving atherogenesis [101,102,103].

4. Macrophage Scavenging and Metabolism of Lipids

Once in the lesion, monocytes encounter macrophage colony-stimulating factor (M-CSF, also referred to as colony-stimulating factor 1 [CSF1]), recognized by their receptor CSF1 receptor, which stimulates their differentiation into macrophages to promote plaque development [104,105,106]. In fact, within the plaque, monocytes are short-lived and readily differentiated into macrophages within 24 h [90]. As part of the lipid-induced dysfunction that drives atherogenesis, oxLDL also induces endothelial expression of M-CSF [99,107]. Although M-CSF can be produced by numerous cell types, conditional knockout studies have identified smooth muscle and endothelial cells, but not monocytes/macrophages, as producers of M-CSF responsible for monocyte differentiation [108]. In turn, these macrophages scavenge cholesterol-rich lipoproteins that have deposited in the arterial wall to become the foam cells characteristic of atherosclerosis [104,109]. Owing to the modification of LDL that occurs within the plaque microenvironment, macrophages depend on expression of scavenger receptors for the uptake of oxLDL. The increased negativity of LDL resulting from its oxidation increases its binding affinity to scavenger receptors, and its retention is supported by binding to proteoglycans, which together increase oxLDL uptake by macrophages and ultimately foam cell genesis within the plaque [110,111,112,113].

The most prevalent of these scavenger receptors responsible for modified lipoprotein uptake is the cluster of differentiation 36 (CD36) and scavenger receptor class A (SR-A). CD36 signals via assembly into a heterotrimeric signaling complex with toll-like receptors 2/4/6 (TLR2/4/6), whereas SR-A signals through the homo-trimeric complex [114,115,116,117,118]. The engagement of these receptors by oxLDL initiates extensive signaling mechanisms that promote the production of pro-inflammatory cytokines, oxLDL uptake, and cell death, all of which increase the pro-inflammatory microenvironment of the plaque [119,120,121]. In addition, macrophages also express LOX-1, which, under basal conditions, accounts for a relatively low amount of oxLDL uptake (~5–10%) but is markedly increased upon stimulation and upregulation of LOX-1 [122]. CD36 was first discovered in human monocytes when precipitated with the OKM5 antibody [123]. Since then, the role of CD36 in atherosclerosis has been controversial. In some studies, CD36 knockout mice showed a decrease in foam cell formation coupled with decreased total cholesterol levels [124,125]. However, other studies have shown the opposite, with CD36 or SR-A knockout mice having increased cholesterol levels with increased or no change in lesion area [126]. Furthermore, double knockout of CD36 and SR-A in mice improves features of atherosclerosis progression, such as death and necrotic area, without impacting overall lesion area or lipid parameters [127]. In addition to differences in numerous variables, these studies serve to underscore the complexity of these receptors apart from their roles in scavenging lipids and the need to better understand the lipid-inflammatory axis in ASCVD. Apart from oxLDL-mediated signaling, TLR4 participates in the catabolism of agLDL for uptake in macrophages in cooperation with CD14 in a MyD88-dependent pathway involving lysosomal exocytosis [128]. Apart from receptor-mediated lipoprotein uptake, a recent study has identified macropinocytosis, a form of fluid-phase uptake of macromolecules mediated by membrane ruffling, as contributing to foam cell development in both murine and human atheromas [129].

Regardless of the route of engulfment, ingested lipids undergo endocytic trafficking to the lysosome for metabolism, where free cholesterol is liberated from the lipoprotein via hydrolysis by lysosomal acid lipase (LAL) [130]. However, free cholesterol is cytotoxic to the cell and must be metabolized or eliminated to preserve cellular homeostasis. In the endoplasmic reticulum (ER), free cholesterol is esterified to fatty acids by Acyl-CoA:cholesterol acyltransferase 1 (ACAT1) to produce cholesteryl esters, which are safely stored in lipid droplets. These lipid droplets can be catabolized by a process known as autophagy to liberate free cholesterol and fatty acids [131]. This free cholesterol can then be removed from the macrophages by cholesterol efflux, which is the first step in RCT [28]. ABCA1 and ABCG1, known as cholesterol efflux transporters, remove free cholesterol to the extracellular compartment via their acceptors, Apo-A1 and HDL, respectively [28,132,133]. In the cell, cholesterol can also be converted to oxysterols by enzymes such as cytochrome P450 enzymes or hydroxylases, or by non-enzymatic means such as reactive oxygen species [134]. Oxysterols such as 22-, 24-, 25-, or 27-hydroxycholesterol serve as endogenous ligands for the liver x receptors α/β (LXRα/β), which have been shown to have anti-atherogenic effects [135,136,137,138,139,140]. Belonging to the nuclear receptor superfamily, LXRs are activated by these oxysterols to act as transcription factors to promote cholesterol homeostasis, including the induction of genes involved in RCT, such as ABCA1, ABCG1, and CYP7A1. Of note, mutations in ABCA1 lead to Tangier disease, a genetic condition resulting in the accumulation of cholesteryl esters in various tissues and low levels of serum HDL [141,142]. Furthermore, LXRs also repress inflammatory gene transcription through antagonism of transcription factors like NF-κB or cis-repression of inflammatory genes [143,144], which together reduce atherogenesis. Additionally, intermediates of cholesterol synthesis, such as desmosterol, also induce signaling in macrophages to dampen inflammation and repress atherosclerosis development [145,146]. In addition to macrophages, a significant proportion of foam cells are derived from smooth muscle cells in both murine and human atheromas [147,148,149,150]. Indeed, several of the aforementioned mechanisms of lipid handling are defective in these foam cell subsets. This includes low levels of LAL for hydrolysis of cholesteryl esters and their limited capacity to engage autophagy of lipid droplets, subsequently impairing cholesterol efflux [151,152].

However, in the setting of excess lipid content within the atherosclerotic plaque, these homeostatic mechanisms go awry, leading to macrophage foam cell dysfunction and their active contribution to the advancement of this disease. One of the hallmarks of foam cells is endoplasmic reticulum stress, including the unfolded protein response (UPR). This consists of PKR-like eukaryotic initiation factor 2A kinase (PERK), activating transcription factor 6 (ATF6), inositol-requiring enzyme 1 (IRE1), as well as activation of CCAAT-enhancer-binding protein homologous protein (CHOP), which together function to promote lipid-induced cell death [104,153,154]. Apart from intracellular cholesterol stores, the formation of cholesterol-rich inflammarafts at the plasma membrane allows for the assembly of inflammatory receptor dimers and engagement of downstream inflammatory signaling [155,156]. Although under steady-state conditions, 25-hydroxycholesterol promotes LXR activation to mitigate intracellular cholesterol stores, in the setting of excess cholesterol accumulation, recent studies show 25-hydroxycholesterol accumulation in human coronary atheromas [157]. Importantly, independent of LXR activation, 25-hydroxycholesterol produced by macrophages increases vascular inflammation through reorganization of membrane cholesterol and TLR4 signaling, in addition to promoting adverse plaque remodeling.

Fatty acids also play critical roles in driving the pathogenesis of atherosclerosis. As previously mentioned, autophagy, which is involved in the hydrolysis of cholesteryl esters, also releases free fatty acids [131]. Fatty acids in macrophages can also be produced by de novo fatty acid synthesis, autophagy of lipid droplets, or can be taken up from the extracellular compartment via scavenger receptors [158,159,160]. In fact, the metabolism of fatty acids has been shown to drive macrophage phenotypes, influencing atherosclerotic plaque pathology. Many studies have shown that the balance between fatty acid oxidation (catabolic) and synthesis (anabolic) induces an anti-inflammatory (M2-like) or pro-inflammatory (M1-like) phenotype of macrophages, respectively, thus influencing overall atherosclerotic plaque pathology [160,161,162,163].

5. Fatty Acids as Signaling Molecules

Apart from serving as metabolic substrates, fatty acids and their derivatives also act as signaling molecules. Fatty acid diversity encompasses carbon chain length and degree of saturation, which can be saturated, monounsaturated, or polyunsaturated. Saturated fatty acids are prevalent and drive chronic inflammatory diseases, including atherosclerosis, via engagement of CD36 and TLRs to promote downstream inflammasome activation [118,160,164]. A prominent example of such a long-chain saturated fatty acid with profound effects on atherosclerosis is palmitate. In addition to metabolism, fatty acids such as palmitate can also serve as substrates for post-translational modifications. In particular, palmitoylation of CD36 and LOX-1 promotes receptor processing and localization to lipid rafts, half-life, and oxLDL uptake to drive foam cell accumulation [165,166].

Additionally, polyunsaturated fatty acids (PUFAs) have been extensively studied in the context of atherosclerosis, in particular omega-3 and omega-6 (ω-3 and ω-6, respectively) PUFAs [167]. A prevalent example of these signaling fatty acid species is arachidonic acid (AA), which is a polyunsaturated omega-6 fatty acid that provides fluidity to cell membranes [168]. Relevant to atherosclerosis, engulfment of oxLDL and subsequent metabolism produces AA in macrophage foam cells [169,170]. AA can be produced via metabolism from linoleic acid, which is an essential ω-6 PUFA that cannot be synthesized by mammals, or by AA release from cell membranes by phospholipases. Regardless of the route of production, AA can be metabolized to produce eicosanoids via enzymatic means, including i) prostaglandins and thromboxanes produced by cyclooxygenases (COX), ii) leukotrienes produced by lipoxygenases, and iii) hydroeicosatetraenoic acids (HETEs) or epoxyeicosatetraenoic acids (EETs) by cytochrome P450 enzymes [168]. These metabolites and enzymes all play varying roles in ASCVD [170].

However, of greater interest to ASCVD are ω-3 PUFAs, which represent another class of bioactive fatty acid species and are synthesized from α-linolenic acid, an essential fatty acid found in vegetable and fish oils, seeds, and nuts [171]. In general, several epidemiologic studies have associated increased consumption of ω-3 PUFAs, such as fish, with a lower risk of CVD [172,173,174]. Among the most relevant ω-3 PUFAs to CVD are eicosapentaenoic acid (EPA), which is synthesized from α-linolenic acid, and docosahexaenoic acid (DHA), which is produced by conversion from EPA. EPA, in particular, has been the subject of several clinical trials wherein the Japan EPA Lipid Intervention Study (JELIS) and the Reduction of Cardiovascular Events with Isocapent Ethyl Intervention Trial (REDUCE-IT) studies have both shown that EPA lowers the risk of CVD events [175,176].

EPA and DHA can be further metabolized into a class of molecules known as specialized pro-resolving mediators (SPMs), including lipoxins, maresins, protectins, and resolvins [177,178]. Here, we will focus on resolvins, which are the SPMs that have been best studied in atherosclerosis. The E-series of resolvins is derived from EPA, of which resolving E1 (RvE1) is the most relevant in atherosclerosis. RvE1 binds to ChemR23, a G protein-coupled receptor (GPCR) whose engagement promotes efferocytosis and dampens macrophage inflammation [179,180]. In contrast, DHA produces maresins, protectins, as well as the D-series of resolvins [177,178]. Resolvin D1 (RvD1), in particular, has been the most well-studied of this series in atherosclerosis. RvD1 binds to a GPR32, a GPCR, and an ALX/FPR2 receptor. Overall, RvD1 and its receptor have also been shown to have atheroprotective roles by promoting efferocytosis and dampening inflammation [178,181,182,183].

6. Other Lipid Species as Drivers of Atherosclerosis

Although cholesterol-rich lipoproteins were initially recognized as conferring risk for ASCVD, recent studies have shown that TRLs are also causal agents of atherosclerosis, including studies demonstrating that TRLs on a per particle basis are more atherogenic than LDL [16,184,185,186]. In addition to Apo-B, TRLs also possess Apo-C3, which in both type 1 and type 2 diabetic patients positively correlates with the risk of developing ASCVD, with loss-of-function mutations associated with a reduction in circulating triglycerides and atherosclerosis [187,188,189,190,191,192,193]. Mechanistic studies have attributed these deleterious roles of Apo-C3 to increased formation of VLDL by the liver and inhibiting lipoprotein lipase-mediated lipolysis of VLDL, thereby blunting its metabolism and maintaining high TRL circulating levels [194,195,196]. Though best studied in the context of TRL, Apo-C3 is also present on LDL and contributes to its atherogenic effects [197].

As indicated in Table 1, lipoprotein (a) (Lp(a)) is another critical lipoprotein species that confers ASCVD risk [198,199]. Lp(a) is a modified form of LDL, where the apolipoprotein(a) glycoprotein is covalently linked to Apo-B100 via a disulfide bond [200,201]. Interestingly, recent genetic evidence using Mendelian randomization demonstrated that Apo-B associated with Lp(a) has a greater hazard ratio for atherosclerosis than LDL-ApoB [202,203]. Lp(a) also contains oxidized phospholipids (discussed below), which account for some of its atherogenic effects [204,205]. In addition, Lp(a) supports leukocyte extravasation into the plaque by interacting with monocyte integrins and stimulating endothelial cell release of chemokines and expression of adhesion molecules to support monocyte-endothelial interactions [206,207]. Indeed, macrophage foam cells can also uptake Lp(a), which promotes apoptosis through CD36-TLR2 signaling [118,208].

Another lipid species that has received decades of attention for its role in atherogenesis is ceramides, which consist of a fatty acid conjugated to sphingosine. The biology of ceramides and their in-depth metabolic pathways have been extensively reviewed elsewhere [209,210], where cellular levels of ceramides are regulated by breakdown from sphingomyelin found in the cell membrane, de novo biosynthesis, or ceramide catabolism. Given the drastic roles this class of sphingolipids plays in disease, ceramides have been used as a clinical biomarker for a variety of diseases, including diabetes and CVD [211]. Owing to the variability in fatty acid species, as described above, in humans, specific ceramide subspecies found in circulation have been associated with increased CVD risk [212,213,214,215,216,217]. Ceramides are found enriched in LDL and therefore can be deposited in the arterial wall. Furthermore, increased circulating levels of these ceramides are associated with increased plaque pathology, including vulnerability and necrotic core content, leading to future MACE [218,219]. In mice, strategies to increase ceramides by targeting metabolism, as described above, have demonstrated an overall pro-atherogenic effect through promoting lipoprotein transport and retention in the plaque, LDL aggregation, and monocyte adhesion [220,221,222,223,224].

In addition to cholesterol, oxidized phospholipids (oxPLs) found on lipoproteins also act as signaling lipids to induce atherogenesis [225]. Of importance, oxPL-ApoB levels show a greater correlation with Lp(a) rather than oxLDL in circulation, likely due to the stronger association of oxPLs in Lp(a) [204,226]. Furthermore, oxPL-ApoB levels show greater predictive value for future CVD as compared to Lp(a) levels, further impressing the importance of oxPL in atherogenesis [205]. oxPLs are a heterogeneous species, where over 30 different oxPLs have been identified in atherosclerotic plaques [227]. Beyond association studies, the pathogenicity of oxPLs was demonstrated in an atherogenic mouse expressing a natural antibody to (E06-scFV), which neutralizes oxPL signaling, resulting in a reduction in circulating lipid levels and overall atherosclerotic plaque area [228]. Neutralizing oxPLs dampens macrophage inflammatory status, apoptosis, and scavenger receptor signaling [118,228,229,230,231]. Two prominent examples of oxPLs investigated in the pathogenesis of atherosclerosis are 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC) and oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (oxPAPC), which accumulate in the plaque to induce cell death in the vessel wall, regulatory T cell dysfunction, and pro-inflammatory responses in monocytes and macrophages [232,233,234,235,236,237,238].

7. Lipids as Drivers of Non-Apoptotic Cell Death

One of the key features of the advanced or vulnerable plaque is the development of a necrotic core, which is an acellular region that develops as cells shift towards pro-inflammatory modes of cell death and ultimately leads to the fatal complications associated with ASCVD [239]. Although best described in the context of inflammation, here we will highlight the lipid-dependent mechanisms by which these processes occur and drive plaque pathology. While necrosis occurs in a signaling-independent manner, necroptosis also results in cell membrane lysis but importantly relies on a cell signaling cascade. This notably involves the phosphorylation of necrosome components, receptor-interacting serine/threonine-protein kinases 1 and 3 (RIPK1 and 3), leading to the activation of mixed lineage kinase domain-like protein (MLKL) and its insertion into the membrane [239]. Necroptosis has been identified as a process that occurs in both mouse and human atheromas and can be engaged by oxLDL [240]. Furthermore, silencing RIPK1, RIPK3, or MLKL or blocking necroptotic signaling have all demonstrated the importance of necroptotic cell death to the development of the necrotic core during atherosclerosis [240,241,242,243]. Importantly, necroptotic cells release lipid mediators, such as prostanoids, that further promote the inflammatory milieu of the plaque in part by blunting the clearance of dead cells [182].

Pyroptotic cell death also involves the formation of pores in the plasma membrane [239]. This form of cell death is characterized by the assembly and activation of the NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome and production of IL-1β, which is released through pores formed by proteins known as gasdermins, particularly gasdermin D [244]. The inflammasome involved in pyroptosis has been identified in human atheromas in the vicinity of the necrotic core and is been strongly associated with ASCVD [245,246,247]. Cholesterol crystals, which accumulate as a result of excess free cholesterol content, are found in the necrotic core and drive lysosomal damage and the activation of the NLRP3 inflammasome [115,248]. Importantly, gasdermin D must be cleaved by caspase-1 to allow for the N-terminal portion to participate in pore formation. However, recent evidence has shown that conjugation of the fatty acid palmitate (S-palmitoylation) to either intact or cleaved gasdermin D at Cys191 also promotes its membrane translocation, although this was shown to be more efficient in cleaved gasdermin D [249,250]. This lipid-dependent activation of the inflammasome, as described here for pyroptosis, also stimulates NETosis, whereby neutrophils release extracellular traps rich in DNA and enzymes [239,251].

Ferroptosis is characterized by lipid peroxide species via the peroxidation of PUFAs that occurs in an iron-dependent manner, which in turn disrupts the plasma membrane, leading to pore formation and cell death [239]. Furthermore, the metabolism of these lipid peroxides can form further lipid species that contribute to cellular toxicity. Interestingly, this form of cell death also contributes to LDL oxidation and thus the pathogenicity of this lipoprotein [252,253]. Indications of ferroptosis in human atheromas include the presence of iron-rich foam cells, hemoglobin from erythrophagocytosis, and ferroptotic regulators ferritin and heme oxygenase 1 (HMOX1) [254,255]. In mice, administration of the ferroptosis inhibitor ferrostatin-1 decreased overall lesion area and cell death, which was demonstrated to be driven in part by oxLDL-mediated lipid peroxidation and endothelial cell dysfunction [256].

8. Lipid Therapies in Atherosclerotic Cardiovascular Disease

Undoubtedly, with the overwhelming evidence of lipids as central drivers of ASCVD, it is unsurprising that targeting dyslipidemia remains an intensively investigated avenue for clinical intervention (Figure 2). Given the primary role of the liver in synthesizing and exporting lipoproteins, many lipid-based strategies for intervening in ASCVD involve targeting the liver. The best example are statins, which remain the most widely prescribed class of drugs in the world [257]. First discovered in 1976 and approved for use in 1987, statins inhibit HMG-CoA reductase, which is the rate-limiting enzyme in cholesterol biosynthesis in the liver, by competitive inhibition [20]. Across numerous studies, statins have been found to lower circulating cholesterol levels, leading to lower all-cause mortality and MACE, as well as being associated with reductions in plaque area and improved plaque stability [258,259,260]. Despite the success of statins, approximately 10–15% of patients develop statin intolerance [261,262]. Therefore, additional avenues of lipid lowering are currently being investigated to improve outcomes. Similar to statins, bempedoic acid has arisen as a recently approved drug to lower cholesterol biosynthesis in the liver [263]. Functioning as an inhibitor of the ATP citrate lyase (ACLY) enzyme, bempedoic acid blocks the conversion of citrate to acetyl-CoA, which serves as the first substrate in the cholesterol biosynthetic pathway. This therefore arrests cholesterol synthesis upstream of HMG-CoA reductase. In patients receiving the maximum tolerated statin dose or those who were statin intolerant, bempedoic acid led to a 16.5–21.1% reduction in LDL cholesterol compared to placebo, which was accompanied by a 13% reduction in MACE [264,265]. Another strategy to lower circulating cholesterol levels has been to target ApoB, which is a critical component of LDL and therefore also reduces LDL cholesterol levels. Mipomersen, an antisense oligonucleotide targeting Apo-B100, was initially approved for human use in patients with FH due to reductions in circulating LDL cholesterol and Lp(a); however, this approval was withdrawn due to hepatotoxicity concerns [266,267,268,269]. A more recent advance for targeting hepatic lipid metabolism is PCSK9, which prevents LDLR recycling to the membrane to support LDL clearance from the circulation, thereby inducing its degradation and promoting sustained hypercholesterolemia [270]. Currently, two monoclonal antibodies (Alirocumab and Evolocumab) and one small interfering RNA (Inclisiran) are approved for clinical use, which show reductions in circulating cholesterol levels and improved hazard ratios for primary cardiovascular endpoints [271,272,273,274,275]. Furthermore, Inclisiran was also shown to improve LDL cholesterol levels in patients with heterozygous FH [276]. In a recent multi-center clinical trial, it was found that Evolocumab reduced the lipid-rich necrotic core in the carotid bifurcation of patients with established but asymptomatic carotid stenosis [277]. Analysis of the ODYSSEY Outcomes clinical trial, which tested Alirocumab in ASCVD, revealed that baseline Lp(a) levels predicted future MACE and that there were beneficial reductions in Lp(a) levels with PCSK9 inhibition [278]. This has sparked multiple phase three clinical trials to test the effect of small interfering RNA and antisense oligonucleotide approaches to disrupting Lp(a) production by the liver on CVD outcomes [279].

Since the initial discovery of LDL cholesterol as a major risk factor of ASCVD, subsequent clinical cohorts have identified elevations in circulating triglyceride levels associated with CVD risk [15]. Based on the mechanisms described above, Apo-C3 has risen as a clinical target to reduce circulating triglyceride levels. Two approved antisense oligonucleotides targeting Apo-C3, Volanesorsen and Olezarsen, have shown reductions in circulating triglycerides, Apo-B, and non-HDL cholesterol [280,281,282]. Other classes of proteins being investigated for their triglyceride-reducing properties are angiopoietin-like proteins 3, 4, and 8 (ANGPTL3, 4, 8), which are LPL inhibitors produced by the liver [283]. Together, these ANGPTLs inhibit metabolism and increase circulating triglyceride levels. Although antibody and gene silencing approaches are being investigated to target these ANGPTLs, only the ANGPTL3 monoclonal antibody Evinacumab has been approved for patients with persistent hypercholesterolemia, such as those with homozygous FH, which decreases circulating LDL cholesterol levels [284,285].

Apart from targeting hepatic production of lipoproteins, another therapeutic strategy is to directly target the lipoproteins that mediate these effects in circulation. Owing to associations between low HDL cholesterol levels and CVD events, as well as the well-known role for HDL in RCT as well as other anti-atherogenic processes, there has been significant interest in efforts to boost HDL as a therapeutic measure to combat ASCVD [286]. However, Mendelian randomization studies to explore the causal effects of HDL on ASCVD, along with a variety of drugs to raise HDL cholesterol, have challenged this “HDL hypothesis” with many of these studies showing null effects on improving cardiovascular outcomes (reviewed in [286]). A prominent example of a class of HDL cholesterol-boosting drugs is cholesteryl ester transfer protein (CETP) inhibitors. CETP is responsible for the transfer of triglycerides from VLDL and LDL to HDL in exchange for cholesteryl esters, effectively raising LDL cholesterol and decreasing HDL cholesterol, thus providing a lipoprotein profile associated with atherogenesis [287,288]. Of the four phase three clinical trials that have investigated CETP inhibition to raise HDL cholesterol, three have failed to show improvements in CVD outcomes. However, one recent trial has shown positive results with respect to risk reduction in the first coronary event during the 4-year follow-up [289]. Newer approaches to address raising HDL in circulation include the administration of recombinant HDL (rHDL) and Apo-A1, which in animal models have shown removal of cholesterol from the plaque, as well as plaque regression (reviewed in [290]). However, the most recent AEGIS-II phase three clinical trial, which investigated the infusion of purified Apo-A1 in patients after acute myocardial infarction [291], did not show improvement in MACE up to 1-year post-treatment.

9. Conclusions

Despite emerging studies that identify additional risk for ASCVD, and even after more than 70 years of intense investigation, it remains undeniable that lipids, as a diverse species, play critical roles in the pathogenesis of the atherosclerotic plaque. Beginning with observations and early therapeutic strategies to target the production and accumulation of lipids, primarily cholesterol, in the arterial wall, a wealth of subsequent studies has identified these lipids as active biomolecules in driving disease progression. Alternatively, significant interest has grown in harnessing the beneficial aspects of lipid biology to promote disease regression. Although statins have proven effective at reducing cholesterol levels, there remains a lipid-dependent influence on ASCVD. Nevertheless, new treatments on the horizon illustrate the benefit of further investigation into lipid-lowering therapies and potential effects on downstream signaling pathways to improve overall cardiovascular health.